Team:SDU-Denmark/labnotes9

From 2010.igem.org

(Difference between revisions)
(Miniprep of PS+B0015 sample Q and pSB3T5 containing J13002)
(Flagella Group)
Line 8: Line 8:
== Flagella Group ==
== Flagella Group ==
 +
 +
=== Two-step PCR of miniprep ===
 +
<br>
 +
==== First step: PCR of miniprep with mutation primers ====
 +
<br>
 +
''Done by:'' Louise <br>
 +
''Date:'' September 7th <br>
 +
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
 +
''Notes:'' <br>
 +
'''3 x Premix 1:''' <br>
 +
114.3ul water <br>
 +
15ul Pfu Buffer <br>
 +
4.5ul dNTP <br>
 +
3ul MgSO4 <br>
 +
4.5ul FlhDC fw <br>
 +
4.5ul FlhDCmut rev <br>
 +
1.2ul PFU <br>
 +
3ul template (miniprep) <br><br>
 +
'''3 x Premix 2:''' <br>
 +
114.3ul water <br>
 +
15ul Pfu Buffer <br>
 +
4.5ul dNTP <br>
 +
3ul MgSO4 <br>
 +
4.5ul FlhDCmut fw <br>
 +
4.5ul FlhDC rev <br>
 +
1.2ul PFU <br>
 +
3ul template (miniprep) <br><br>
 +
''Results:''
 +
<br>
 +
NanoDrop: <br>
 +
'''Sample 1.1:''' Concentration: 359.5ng/ul Purity: 1.83/2.20 <br>
 +
'''Sample 2.1:''' Concentration: 304.1ng/ul Purity: 1.85/2.23 <br>
 +
<br>
 +
The PCR samples were run on a 1.5% gel with a 100bp-1000bp ladder. The first three samples are all positioned between 100bp and 200bp, this is the part of the gene run with FlhDCmut fv and FlhDC rev primers. The other three samples are all positioned betveen 900bp and 1000bp, this is the pcr with FlhDC fv and FlhDCmut rev primers. All samples looked okay and were pooled as sample 1.1 (light band) and sample 2.1 (heavy band)<br>
 +
[[Image:Team SDU-Denmark-Pfx pcr af miniprep.jpg|300px]]
 +
<br>
 +
A gel extraction of the PCR product of sample 1.1 and 2.1 were made:
 +
<br>
 +
[[Image:Team SDU-Denmark Gel extraction af FlhDCmut dele.jpg|300px]]
 +
<br>
 +
The gel extraction products were used in the preceding PCRs.
 +
 +
==== Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers ====
 +
<br>
 +
''Done by:'' Maria <br>
 +
''Date:'' September 9th <br>
 +
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
 +
''Notes:'' <br>
 +
'''Premix:''' <br>
 +
38ul water <br>
 +
5ul PFU buffer + MgSO4 <br>
 +
1.5ul dNTP <br>
 +
1ul Sample 1.1 <br>
 +
1ul Sample 2.1 <br>
 +
1.5ul FlhDC fw primer <br>
 +
1.5ul FlhDC rev primer <br>
 +
0.5ul PFU <br><br>
 +
'''PCR Program:''' <br>
 +
<table style="text-align: left; height: 260px; width: 225px;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">1:Start<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">95C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">2: Denaturing<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">95C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">30 sec<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">3: Annealing<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">56C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">30 sec<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">4: Elongation<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">72C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">5:<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">GO TO<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 rep. 4x<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">6: Denaturing<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">95C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">30 sec<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">7: Annealing<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">63C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">30 sec<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">8: Elongation<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">72C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">9:<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">GO TO<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">6 rep. 25x<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">10: End <br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">72C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">5 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">12: Hold<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">4C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;"><br>
 +
</td>
 +
</tr>
 +
</table> <br><br>
 +
 +
''Results:'' <br>
 +
Some of the PCR product was run on a 1.5% gel with a 10kb ladder. <br>
 +
[[Image:Team SDU-Denmark God pcr af FlhDCmut (maria) 1.jpg|300px]]
 +
<br>
 +
The rest of the product was extracted from a new gel, unfortunately there were problems with the camera, so there is no picture of the gel extraction.
 +
<br>
 +
===== PCR of Gel extraction =====
 +
<br>
 +
''Done by:'' Maria <br>
 +
''Date:'' September 9th <br>
 +
''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
 +
''Notes:'' <br>
 +
'''Premix x 6:''' <br>
 +
234ul water <br>
 +
30ul PFU buffer + MgSO4 <br>
 +
9ul dNTP <br>
 +
9ul FlhDC fw primer <br>
 +
9ul FlhDC rev primer <br>
 +
2.5ul PFU <br>
 +
6ul template <br>
 +
 +
<table style="text-align: left; height: 260px; width: 225px;" border="1"
 +
cellpadding="2" cellspacing="2">
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">1:Start<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">95C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">2: Denaturing<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">95C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">30sec<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">3: Annealing<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">63C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">30 sec<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">4: Elongation<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">72C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">5:<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">GO TO<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">2 rep. 29x<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">6: End <br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">72C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;">5 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top; width: 102px;">7: Hold<br>
 +
</td>
 +
<td style="vertical-align: top; width: 52px;">4C<br>
 +
</td>
 +
<td style="vertical-align: top; width: 45px;"><br>
 +
</td>
 +
</tr>
 +
</table> <br><br>
 +
<br>
 +
<br>
 +
''Results:''
 +
<br>
 +
The PCR worked fine, unfortunately there were problems with the camera, so there is no picture of the gel. <br>
 +
=== Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3 ===
=== Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3 ===
Line 177: Line 413:
'''Results:''' <Br>
'''Results:''' <Br>
<Br><Br>
<Br><Br>
-
 
== Photosensor group ==
== Photosensor group ==

Revision as of 09:47, 1 October 2010